This disclosed technology relates to stimulated emission fluorescence and stimulated Raman microscopy and more particularly to in-vivo stimulated emission.
There is interest in providing deep imaging for use in research, neuroscience, endoscopy, dermatology and intra-surgical definition of clear margins during removal of malignant tissues. For example, Optical Coherence Tomography (OCT) can obtain images up to 1 mm depth in tissue.
Multi-Photon Excitation (MPE) imaging can enhance the depth of penetration by using infrared photons for excitation where tissue absorption is low. MPE uses two or more photons to excite emission as shown in FIG. 1b. In MPE, the two or more photons can be simultaneously absorbed by one molecule, through the population of one, or more, very short lived virtual states.
MPE excitation has also been used in Fluorescence Lifetime Microscopy (FLIM), for example, to measure the fluorescent lifetimes of bound and free state metabolic cofactor NADH. Fluorescent lifetimes are of importance when determining a metabolic state of cells, in accessing tissue health and differentiating normal from malignant cells. MPE, however, is relatively slow because the fluorescent yield of free NADH is low, has a short excited state lifetime and needs photon counting to create a decay curve.
Standard fluorescence is an incoherent spontaneous emission process where emission of one or multiple photons causes fluorescent emission. In standard fluorescence, the incoherent spontaneous emission can be red shifted from the excitation and can be considered a dark field imaging technique. The measurement process for standard fluorescence is limited to background fluorescence and electrical noise.
Stimulated fluorescent emission (STEM) imaging is a combination of incoherent and coherent processes. (the energetics of which are shown in FIG. 1a) that uses two photons—a pump and a probe. The pump excites an electron into excited state S1 from ground state S0. A several hundred femtosecond delay, or more, is allowed for the decay of an excited state vibrational level into the lowest excitation level in the excited state manifold S2 via a Kasha decay process. Then a probe (or stimulated emission) beam causes the stimulated emission of a photon and the de-excitation of the electron to S3, which then rapidly decays via a Kasha decay process back to S0. The signal measured is a gain in the probe beam or loss in the pump beam. STEM techniques have been used to image molecules that absorb strongly, but do not fluoresce efficiently such as oxy-hemoglobin, deoxy-hemoglobin, melanin, cytochromes and certain drugs.
STEM is a bright field technique where a signal is added to the forward propagating probe beam. The gain in the beam is 10−4-10−7 (depending on concentration). Therefore, sophisticated electronic signal processing lock-in techniques are usually required to detect a small probe beam change. STEM imaging also uses moderate to high concentrations of molecules to image tissue at moderate to high speed. Unlike fluorescence imaging where emission occurs in any direction, the emission in STEM occurs in the forward direction. Therefore multiple scattering events are required to collect the signal at the tissue surface. STEM is best used for weakly absorbing and scattering tissues but the depth of imaging is limited and requires collection at an angle outside of the imaging aperture, eliminating the ability to do confocal imaging and degrading signal to noise ratio by collecting photons that scatter prior to reaching focus.
Multi-Photon Stimulated Emission Microscopy (MP-STEM) can be used to enhance the depth of penetration and reduce the scattering and absorption of stimulated emission photons in STEM microscopy. MP-STEM uses multiple photons for both excitation and to stimulate emission from weakly fluorescent molecules. The process of MP-STEM can reduce the focal spot size of the emitting region. When using 3 photons or more, the focal spot is reduced in size enough to cause the stimulated emission spot to be small enough to cause dipole-like backscatter emission. This occurs when the axial dimension of the emitting region shrinks to less than 50% of the stimulated emission wavelength. Dipole backscatter enables enhancement of the detected Signal to Noise Ratio (SNR) because in the back scattered direction the noise is due to the Refractive Index (RI) gradient and MIE scattering from the emission region focus in confocal microscope geometry. This is less than the forward scattered noise normally detected in STEM microscopy, or multiple backscatter STEM detection.
There are deficiencies in the use of MP-STEM.                1. It is a single point scanning system and therefore image throughput can be low. This is especially the case when it is desired to perform a 3D reconstruction of tissue being sampled.        2. The focal spot shrinkage in 2 and 3 photon MP-STEM is not small enough to provide optimal dipole backscatter.        3. Optimal focal spot reduction uses high Numerical Aperture (NA) imaging. This, typically, does not enable large standoff distances that can be desirable for in-vivo imaging.        
The throughput in single point scanning MPSTEM imaging can be similar to that encountered in single point confocal fluorescence imaging. In fluorescence imaging applications Light Sheet Fluorescence Microscopy (LSFM) and Structured Illumination Microscopy (SIM) can be used to increase the throughput in imaging. Multiple image points are collected at one time and computer reconstruction can be used to rapidly create a final image. The embodiments of these techniques typically do not directly transfer to STEM microscopy because two rather than one wavelength must be used in STEM applications and STEM is a bright field technique with a high background and LSFM and SIM are dark field imaging techniques with much lower background noise.
Another stimulated emission technique that could benefit from higher throughput and lower background noise is Stimulated Raman Scattering Microscopy (SRSM). This uses stimulated vibrational transitions, rather than stimulated electronic transitions. The coherent Raman imaging techniques of Coherent Anti-Stokes Raman scattering (CARS) and SRSM have been investigated in this regard because of the ability to use intrinsic Raman vibrational signatures as label-free contrast.
Recently SRSM imaging has been further developed because of certain advantages over CARS imaging. It is substantially free from the non-resonant background present in CARS microscopy. Unlike CARS, the SRS spectrum is substantially identical to standard Raman scattering; it has shot-noise-limited sensitivity; has linear concentration dependence; has an absence of spatial coherence; and has a calculable point spread function. In the non-resonant form, it has limited susceptibility to background fluorescence.
SRSM imaging has been shown to produce images of unstained in-vitro tissue samples with similar structural identification and contrast to that achieved with standard haematoxylin and eosin tissue stains by using CH2 and CH3 vibrations of lipids and proteins. Volume stimulated Raman emission from a scanning microscope occurs in the forward scattered direction, requiring multiple scattering events to direct the light out of the tissues. Back scattered in-vivo images have been obtained with broad area detection to collect the multiply scattered photons. High resolution images with good depth resolution have not yet been shown with this approach. In addition almost all SRSM techniques use either forward scattering or multiple back scattered photons. In addition all previous techniques use single point scanning which slows down the acquisition of 3-D images.